组织工程学黄韧带干细胞的筛选与鉴定

Selection and identification of tissue engineering human ligamentum flavum-derived stem cells

目的：通过Fibronectin介导进行差别粘附法筛选出人黄韧带干细胞并进行鉴定,探讨其作为组织工程种子细胞的可行性。方法：从椎间孔镜或椎间盘镜微创手术中获取10例人黄韧带标本,机械-酶消化法联合获取原代细胞,扩增第二代后接种于Fibronectin包被过的培养瓶。流式细胞仪检测细胞表面特异性标志物。免疫组化方法测定干细胞表达相关特异性蛋白。使用干细胞诱导培养基（实验组）向成骨、成脂、成软骨多向诱导分化,使用常规生长培养基培养者作为阴性对照（对照组）,分别行茜素红染色、油红O染色、阿利辛兰染色鉴定。PT-PCR检测成骨、成脂肪、成软骨基因表达。取同一患者的髂骨骨髓提取骨髓间充质干细胞,统计学分析黄韧带干细胞与骨髓间充质干细胞的细胞周期、增殖能力和干性基因表达差异。结果：原代细胞形态呈三角形或多角形,经Fibronectin介导粘附培养后形态比较均一,呈长梭形克隆群。流式细胞仪检测筛选后的黄韧带干细胞CD90、CD73阳性率〉96.8%,CD105阳性率〉95.9%,而stro-1为阴性。实验组茜素红染色呈强阳性,油红O染色可见大小不等脂滴呈红色,阿利辛兰染色大量蛋白聚糖聚集呈蓝色,对照组均为阴性。PT-PCR测定实验组成骨基因（OC、ALP、RUNX-2）、成脂肪基因（LPL、APP、PPAR2）、成软骨基因（COLⅡ、AGG、SOX9）显著上调,其中RUNX-2在对照组有低水平表达。免疫组化显示黄韧带干细胞和骨髓间充质干细胞均表达a-SMA、Ⅰ型胶原、纤连蛋白,但均不表达Ⅱ型胶原。两种干细胞均超过80%的细胞比例处于G0/G1期提示都有很强自我更新能力（P〉0.05）。CCK-8检测不同时间点两种细胞的增殖能力,在第1~10天时OD值逐渐增加,第10~14天达稳定状态,二者有相似的增殖能力（P〉0.05）。两种干细胞均表达干性基因NONAG、OCT-4、SOX2,组间差异无显著性（P〉0.05）。结论：通过Fibronectin差别粘附筛选法可有效筛选纯化出黄韧带干细胞,其可向成骨、成脂、成软骨多向分化,为组织工程技术治疗退变椎间盘提供了新的种子细胞。

Objectives： To select ligamentum flavum-derived stem cells by fibronectin differential adhesion assay, and to determine whether MSCs exist in the human ligamentum flavum which provide cell candidates for cell-based regenerative medicine and tissue engineering. Methods： The samples of ligamentum flavum were collected from transforaminal endoscopic surgery or microendoscopic discectomy surgery for lumbar de-generative disease, and primary ligamentum flavum was obtained by mechanical method combined with colla-genas Ⅰ. Primitive cells proliferating to passage 2 were transferred to culture flasks coated with fibronectin.The cells′ special markers were detected by flow cytometry. Cell clusters were analyzed by Immunohistochem-ical Staining for expression of stem cell-related proteins and were induced to differentiate into osteoblasts,chondrocytes and adipocytes under differentiation medium（experimental group）. Negative control wells were maintained in basal Medium. The two groups were stained with alizarin red staining, oil red O and alcian blue respectively. RT-PCR was used to analyze the m RNA expressions of osteogenic, adipogenic- and chon-drogenic-related genes. Human BM-MSCs were isolated from bone marrow aspirates taken from the iliac crest of the same patient. Statistical analysis was used to test the proliferation capacity, cell cycle and stem cell gene expression, and the results were compared with bone marrow MSCs（BM-MSCs）. Results： The primary cell morphology was triangle and multiangualr, but the cells became comparatively uniformly fibroblastic-like.The percentages of CD73 and CD90 positive cells were more than 96.8%, while the percentage of CD1051-positive cells was more than 95.9%, but negative for stro-1. Osteogenic differentiation group was strongly positive by alizarin red staining. In the adipogenic assay, it exhibited characteristic intracellular lipid droplets that could be stained with Oil Red O. In the chondrogenic micromass culture medium, chondrogenic induction micromass formed a sulfated proteoglycan-rich extracellular matrix which exhibited a positive alcian blue staining, while the non-induction group was negative. The m RNA analysis of RT-PCR results revealed that the osteogenesis-related genes（ALP, OC, RUNX-2）, the adipogenesis-related genes（PPAR-2, APP, LPL） and chondrogenesis-related genes（AGG, COL Ⅱ, SOX-9） all markedly increased in induced group compared with the control（non-induction, growth） group（P〉0.05）, interestingly, the low-level expression of RUNX-2 was detected in the non-induction group. Immunocytochemistry staining further confirmed that all LFSCs and BMMSCs expressed type Ⅰ a-SMA, fibronectin, collagen-Ⅰ, but no collagen-Ⅱ. The cell cycle showed remarkably capable of self-renewal of more than 80% of the BMMSCs and LFSCs were in the G0/G1 phase. The OD value increased from day 1 to day 10 and reached a plateau from day 10 to day 14（P〉0.05）, both stem cell types exhibited similar growth tendencies. The stem cell gene（NONAG, OCT-4, SOX2） expression in the LFSCs was similar to that in BM-MSCs and the differences were not significant（P〉0.05）. Conclusions： The ligamentum flavum-derived stem cells obtained by fibronectin differential adhesion assay have capacity for osteogenic, adipogenic and chondrogenic differentiation, which can be used as a new candidate for cell-based regenerative medicine and tissue engineering.